Molecular Basis of Recessively Inherited Eye Disorders in Pakistani Population

Abstract

Eye disorders can occur due to genetic variations that can be inherited. Autosomal recessive eye disorders are considered rare, but there prevalence increases significantly in regions where consanguineous marriages are common. The current study was designed to investigate the molecular basis of recessively inherited eye disorders in Pakistani population. After approval of this study from Bioethical Committee of Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad Pakistan, various hospitals in different cities were visited and samples were collected from twenty primary congenital glaucoma (PCG) families, thirty-five retinal dystrophy families and one microphthalmia family with the help of ophthalmologists. Whole genomic DNA was extracted from each sample and Sanger’s sequencing was carried out for CYP1B1 gene in PCG patients while retinal dystrophy and microphthalmia samples were sent for next generation sequencing to screen for pathogenicity. The identified variants were checked for segregation in family members. Various in-silico tools such as PROVEAN, SIFT, Mutation taster, CADD, REVEL, PolyPhen-2, PANTHER and MutPred were used to analyze the pathogenicity of identified variants. In twenty consanguineous PCG families Sanger sequencing of CYP1B1 gene revealed nine disease causing variants included five missense variants c.457C>G, c.724G>C, c.516C>A, c.740T>A, c.1263T>A, two frameshift variants c.758-759insA, c.789dup and two silent variants c.1314G>A, c.771T>G. Out of these variants, six heterozygous and three homozygous variants were not reported before in any study. During molecular screening of these twenty families seven polymorphisms c.1347T>C, c.1294G>C, c.2244_2245insT, c.1358A>G, c.355G>T, c.142C>G and g.35710_35711insT were also identified one of which was not reported before (g.35710_35711insT). In thirty five retinal dystrophy families’ disease causing variant were identified in eighteen families by next generation panel sequencing and whole exome sequencing for four families. The eighteen variants identified in current study were c.842G>A in AGBL5 gene, c.847C>T in CERKL gene, c.1429C>T in GPR179 gene, c.874C>T in SAG gene, c.559T>C in RHO gene, c.281T>C in ARL6 gene, c.1252T>C, c.1728del and c.1459T>C in CRB1 gene, c.413-1G>A in CNGB1 gene, c.187C>T and c.1560C>A in USH2A gene, c.547C>T in NMNAT1 gene, c.5571_5576delins x CTAGAT in EYS gene, chr2:73775682-73814328 in ALSM gene, c.109del in PAX6 gene, c.471dup in SPATA7 gene, c.1163A>G in ACOX3 gene. Four variants c.1728del (p.Asp576GlufsTer20) in CRB1 gene, c.5571_5576delinsCTAGAT (p. Leu1858*) in EYS gene, c.471dup (p. Pro158Alafs*39) in SPATA7 gene and c.1163A>G (p.Asp388Gly) in ACOX3 gene are not reported earlier in any study. Three of these novel variants were homozygous except for c.1728del (p.Asp576GlufsTer20) that was found in heterozygous condition. One novel variant p. Pro158Alafs*39 was identified in two families RP 112 and RP 113. In family RP 073 a novel homozygous variant c.388C>T; p.Arg130Cys was identified in S1PR2 gene in three deaf and dumb family members who were found normal for retinal disorder. In congenital bilateral microphthalmia family, a previously reported stop gain variant c.720C>A; p. Cys240* was identified in FOXE3 gene during panel capture sequencing. This variant leads to early truncation of protein that cause developmental defects in size of eye socket and was segregated in recessive manner. The stop gain variant affected the exonic splicing enhancer sites and exonic splicing silencer sites and was found to be 100% conserved in similar species. This study provides insight into the highly heterogeneous spectrum of eye disorders in consanguineous families from Pakistan. Genetic counseling was provided to all enrolled families. In PCG families, identification of disease-causing variant in CYP1B1 gene in 45% recruited families draw attention to the involvement of non- coding region of CYP1B1 gene and other genes like LTBP2, TEK, and MYO7A in our population. Identification of different gene mutations in eighteen inherited retinal dystrophy families and no variant in remaining seventeen recruited families stresses on need of whole genome sequencing of these families. Our findings necessitate effective screening of inherited eye disorders in Pakistani families using state of art technologies to reduce disease incidence in upcoming future through genetic counseling to refrain from practicing consanguinity and performing premarital screening as a control measure in upcoming generations.